Fibrinolysokinases from streptomyces

ABSTRACT

Fibrinolysokinase of good stability is isolated from culture solutions of actinomycetes by heat treatment at a low pH followed by fractional precipitation.

U United States Patent [151 3,669,239 Frommer et al. [4 1 May 2, B972 [54] F IBRINOLYSOKINASES FROM [56] References Cited STREPTOMYCES UNITED STATES PATENTS [72] Inventors: Werner Frommer; Otto Wagner, both of 3 140 984 7/1964 Tosoni at al 195/66 B wuppemlElberfeld' Germany 3,444,045 5/1969 Derenzo et al. .....l95/66 B [73] Assignee: Farbenfabriken Bayer Akliengesellschaft, 3,477,913 11/1969 Sloane ..l95/66 B Leverkusen Germany OTHER PUBLICATIONS d: 1 969 [22] He Nov Chemical Abstracts Vol. 69, i968 104046K 2| 1 Appl. No.: 877,818

Primary Examiner-Lionel M. Shapiro [30] Foreign Application Priority Data Anomey Mccanhy Depaoh & O Bnen Nov. 22, 1968 Germany ..P 18 10 277.7 ABSTRACT Fibrinolysokinase of good stability is isolated from culture [52] us. Cl ..l95/66 B Solutions of aminomycetes by heat treatment at a low PH M 511 lm. Cl. ..c12d13 10 lowed byfmctionalprecipitation [58] Field of Search ..l95/62, 65, 66

5 Claims, No Drawings FIBRINOLYSOKINASES FROM STREPTOMYCES BACKGROUND or THE INVENTION 1. Field of the Invention This invention relates to the isolation of a fibrinolysokinase of good stability from culture solutions containing the same.

2. Description of the Prior Art It is known that a fibrinolysokinase, urokinase, can be isolated from human urine. This process of isolation is however expensive because of the difficulty in recovery from the particular starting material used. Furthennore the content of kinases is so low that several 100 liters of urine have to be worked-up for one therapeutic dose [Astrup and Sterndorff, Nature, 170,981 (1952)].

It is further known that a fibrinolysokinase, which is described as streptokinase, can be isolated from haemolytic streptococci. The disadvantages of this substance for medical use reside above all in its high antigenic action and in the requisite expensive purification operation, which increase the price very greatly [Markus and Ambrus, J. Biol. Chem. 235, 1673 (1960) 1.

Further, fibrinolysokinases are known from Bacillus subtilis [Shimi and Kelada, Arch. Mikrobiol. 50, No. 4, 326 (1965) and from Pasteurella pestis [Beesley et al, J. Bacteriol. 94(1), 19 (1967) Here again a practical use is, for known reasons, not possible.

Therefore, to the present time, the art is not cognizant of procedures by which highly stable fibrinolysokinase can be isolated in a simple and efficient manner.

SUMMARY OF THE INVENTION It is accordingly one object of the present invention to provide a procedure for the isolation of a fibrinolysokinase of extraordinary stability.

A further object is to provide a procedure for the isolation of highly stable fibrinolysokinase from culture solutions of actinomycetes.

Other objects of the present invention will become apparent as the description thereof proceeds.

In satisfaction of the foregoing objects and advantages, there is provided by this invention a process for the isolation of a fibrinolysokinase of extraordinary chemical stability in a simple manner from culture solutions of actinomycetes. This isolation is conducted by a heat treatment of the culture at a low pH, for example, about 1.5, followed by fractional precipitation. The molecular weight of this substance is relatively low so that no antigen actions are to be expected.

DESCRIPTION OF PREFERRED EMBODIMENTS As pointed out above, the process of the invention is concerned with a process for the isolation of a fibrinolysokinase, urokinase, a known substance, from culture solutions of actinomycetes. The resulting product is of extraordinary chemical stability.

The process comprises culturing the culture solutions, as described hereinafter, and then separating off the proteases after 1 to 10 days, preferably about 2 to 3 days growth. Thereafter the resulting culture is adjusted to a pH on the acid side, about to 4.0 but preferably about 1.5 with an acid substance such as very dilute acids (e.g. 5 percent to 15 percent aqueous solutions). Acids which may be employed include inorganic acids such as aqueous hydrochloric acid, phosphoric acid, etc., organic acids as well as non-toxic salts which will effect the acidification. A preferred acidification reagent is percent aqueous hydrochloric acid.

After acidification, the resulting mixture is heated at a temperature of about 50 80C., preferably 65C., for about 5 minutes to 1 hour, depending on the temperature. For example, heating at about 65C., requires about minutes.

On conclusion of the heating step, the mixture is immediately cooled and the pH of the mixture then adjusted to a pH of about 5 to 10, preferably 7.5. Adjustment of the mixture to a neutral or. basic pH may be accomplished by the addition of a base such as dilute (5 to 15 percent) NaOH, KOH, LiOH and mixtures thereof as well as alkali metal and alkaline earth metal carbonates, bicarbonates, etc., as well as any other known neutralization substance.

The resulting mixture is then subjected to concentration by centrifuging to remove the liquid or by evaporation of the liquid as by use of a vacuum. Thereafter, fractional precipitation is effected by adding the resulting supernatant liquid to an excess (e.g., a 2- to 6-fold volume) of an organic liquid. Acetone is preferably employed as the organic liquid but other organic liquids such as homologous ketones, ethers, and the like may also be used as this is a common expedient in the art. The mixture is then agitated for a short period at a low temperature, e.g., -40C. to 0C. and then allowed to stand at a temperature of about l0 to +10C. for l to 5 hours. The supematant liquid is then decanted.

The resulting sediment is then taken up again in cold organic solvent with thorough dispersion of the sediment. The

.mixture is then again centrifuged and the resulting sediment worked up one or more times with organic solvent and/or dried in vacuo. Yields of the product will range from about 50 to percent with enrichment being as high as 10fold.

Strains of actinomycetes which produce lysokinases can be easily found in accordance with the method given below. They occur in large numbers among the actinomycetes class.

Strains of the order Actinomycetales especially those of the family of the Streptomycetaceae, and among these, especially strains of the genus Streptomyces, because of their ease of culture, are isolated from samples of soil in a known manner. Culture flasks containing nutrient solutions which permit the growth of these strains are inoculated with samples of these strains obtained by inoculation. It is for example possible to use the glycerine-glycine nutrient solution according to von Plotho, of which the composition is 2 percent glycerine, 0.25 percent glycine, 0.1 percent NaCl, 0.1 percent K HPO,, 0.01 percent FeSO, 7H O, 0.01 percent MgSO 7H O and 0.01 percent CaCO To produce more rapid growth, it is appropriate to further add complex sources of carbon, such as for example corn steep liquor (preferably in concentrations of about 0.25 percent) or soya flour (preferably again in concentrations of 0.25 percent), or both, to such a nutrient solution. In such cases the pH-value of the solution must be adjusted. An initial pH of the nutrient solution of between 6.0 and 8.0, especially between 6.5 and 7.5, is preferred.

The glycerine of the nutrient solution can also be replaced by other sources of carbon such as for example glucose or sucrose. Instead of the glycine, other nitrogen sources such as for example alanine, ethylamine or lysine, can also be used. The concentrations of the carbon sources and nitrogen sources, and the concentration of the salts, may vary within wide limits. FeSO CaCO and MgSO, can also be entirely absent.

In one embodiment ml. of the nutrient solution are filled into l-liter Erlenmeyer flasks, sterilized in a known manner, inoculated with the strain to be investigated, and the flask incubated at 15 to 35C., preferably at 23 to 30C., on shaking machines. When the culture shows growth, which generally occurs after 1 to 10 days, in most cases after 3 to 5 days, a sample of, for example, 5 m1. is taken, the mycelium in this sample is removed by filtration or centrifugation, and the solution is adjusted to a pH of 0 to 4 with approximately 1 I10 HCl. Thereafter the mixture is heated to 65C. for 15 minutes, immediately cooled, and the pH-value adjusted to 5-l0 with approximately l/l0 NaOH. The fibrinolysokinase activity is deten'nined in the usual manner on the Miillertz-Astrup plate. Any residual proteolytic activity is determined in a known manner, again on the heated Miillertz-Astrup plate.

In this invention, there has been tested a whole series of strains of the most diverse genera of the order Actinomycetales in accordance with the method described above and found lysokinase activities which, even though in part weak, were nevertheless distinct, in various genera. The strains of the family of the Streptomycetaceae, especially those of the genus Streptomyces, were the most favorable as regards yield and ease of culture. Of 32 streptomycetes which were tested, the culture solutions showed degradation zones on the non-heated fibrin plates in the case of 16 streptomycetes even if the pl-l-value of the culture solution was adjusted at 1.5-1.8. Of these 16 strains, 6 showed no degradation zones on the heated plate, that is to say with denatured plasminogen, and thus do not contain any proteases after the preceding treatment. In these cases the action must thus be ascribed to a lysokinase.

These 6 strains (for a description of the strains see Table l which on the basis of their sporophore structure all belong to the strain Streptomyces, show the important systematic characteristics listed in Table 1 [Hutter, R., Systematik der Streptomyceten" (Systematisation of the Streptomycetes), Bibliotheca Microbiologica, Fasc. 6, S. Karger, Basel, New York, 1967].

The strains St 16, St 18 and St 24 were deposited in the Centraalbureau voor Schimmelcultures under Numbers CBS 670.68, 671,68 and 669,68.

In order to obtain fibrinolysokinase, these strains are cultured as described above. After 2 to 3 days growth the myceliurn is separated off and the lysokinase enriched from the culture broth, by concentrating the solution and precipitation with organic solvents as described herein.

The following examples illustrate the process of the inventlon.

EXAMPLE 1 A 1 liter Erlenmeyer flask which contains 100 ml. of a nutrient solution of composition 2 percent glycerine, 0.05 percent glycine, 0.4 percent corn steep liquor, 0.4 percent soya flour, 0.1 percent NaCl, 0.1 percent K l-lPO,, 0.01 percent MgSO 7H O, 0.01 percent FeSO 71-1 and 0.01 percent CaCO (sterilization for 60 minutes at 121C.) is inoculated with 1 ml. of a spore suspension which is obtained by rinsing a 10 day old inclined test tube of the strain St 16 together with glycerine-glycine agar, according to von Plotho, with 12 ml. of water. The culture is incubated on a shaking machine at 24C. After 3 days incubation a culture solution is obtained which contains 1.9 CTA units/ml. The almost clear centrifuged filtrate is adjusted to pH 1.5 and kept for 15 minutes at 65C. It is immediately cooled, adjusted to pH 7.5 and again centrifuged. The sediment is discarded. The supernatant liquid is added to a 4-fold volume of acetone cooled to 20C. and the mixture is stirred for minutes. The mixture is then left to stand for 3 hours at +4C. The supernatant liquid is finally decanted and discarded.

The sediment is again taken up in cold dry acetone and thoroughly dispersed therein. Thereafter the mixture is centrifuged and the resulting sediment is dried in vacuo. Yield is about 50 to 80 percent; enrichment, about -fold.

The strain St 16 shows the species characteristics listed in Tables 1, 2 and 3 and is probably to be classified with Streptomyces chrysomallus Lindenbein [Lindenbein, W., Arch. Mikrobiol. 17, 361 (1952) EXAMPLE 2 If a nutrient solution according to Example 1 is inoculated with 1 ml. of a spore suspension which is obtained by rinsing a 10 day old inclined test tube of strain St 24 with 12 ml. of water, and incubated according to Example 1, then a culture solution is obtained after 2 days culture which contains 0.51 CTA units/ml.

The almost clear centrifuged filtrate is for example adjusted to pH 1.5 and kept for minutes at 65C. After cooling immediately, it is adjusted to pH 7.5 and again centrifuged. The sediment is discarded.

The supernatant liquid is added to a 4-fold volume of acetone cooled to C. The mixture is stirred for 5 minutes and is then left standing for 3 hours at +4C. The supernatant liquid is finally decanted and discarded.

The sediment is again taken up in cold dry acetone and thoroughly dispersed therein. Thereafter the mixture is centrifuged and the resulting sediment is dried in vacuo. Yield about 50 to percent. Enrichment, about IO-fold.

The strain St 24 shows the species characteristics listed in Tables 1, 2 and 3 and is probably to be classified with Streptomyces griseus Waksman et Enricci [Waksman, S.A., The Actinomycetes Vol. 11, The Williams and Wilkins Comp., Baltimore(l96l) EXAMPLE 3 If 100 ml. of a nutrient solution containing 3 percent glycerine, 0.4 percent glycine, 0.25 percent corn steep liquor, 0.4 percent soya flour, 0.1 percent NaCl, 0.1 percent K HPO 0.01 percent MgSO. 71-1 0, 0.01 percent FeSO 7H O and 0.01 percent CaCO, (sterilization for 60 minutes at 121C.) are inoculated in accordance with Example 2 with the strain St 24, then a nutrient solution is obtained after 3 days culture which contains 10.2 CT A units/ml.

The further working-up takes place as stated in Examples 1 and 2.

EXAMPLE 4 if 100 ml. of a nutrient solution according to Example 3 are inoculated with 1 ml. of a spore suspension obtained by rinsing a 10 day old inclined test tube of strain ST 18 together with glycerine-glycine-agar according to von Plotho, then a culture broth containing 5.3 CTA units/ml. is obtained after 4 days culture on a shaking machine at 24C.

The further working-up takes place as stated in Examples 1 and 2.

The strain St 18 shows the species characteristics quoted in Tables 1, 2 and 3 and is probably to be classified with Streptomyces chrysomallus Lindenbein [Lindenbein, W., Arch. Mikrobiol. 17, 361 (1952) EXAMPLE 5 EXAMPLE 6 If nutrient solutions which in addition to 2 percent glycerine, 0.4 percent glycine, 0.4 percent soya flour and 0.25 percent corn steep liquor contain the salt concentrations quoted in Table 4 are inoculated according to Example 4, the yields quoted in Table 4 are obtained.

The further working-up takes place as stated in Examples 1 and 2.

EXAMPLE 7 If nutrient solutions of composition 2 percent glycerine, 0.25 percent corn steep liquor, 0.25 percent soya flour, 0.1 percent NaCl, 0.1 percent K HPO 0.01 percent MgSO 7H O, 0.01 percent FeSO 7H O and 0.01 percent CaCO; (sterilization for 60 minutes at 121C) are inoculated, with the addition of various aminoacids in concentrations of 0.4 percent according to Example 4, then the following yields are obtained after 4 days culture at 24C:

Addition (0.4%) CT A Units/ml.

Alanine 6.2 L-Tyrosine 0.3 1

L-Lysine 0.39 Duration of Culture CT A Units/ml. Glycine 4.8 (hours) 60 7.0 The further working-up takes place as stated in Examples 1 5 63 and 2. 76 8.4

EXAMPLE 8 If a fermenter containing 60 liters of a nutrient solution of 2 1 0 further workmg'up takes place as Stated m Examples l percent glycerine, 0.4 percent glycine, 0.25 percent corn steep liquor, 0.4 percent soya flour, 0.1 percent NaCl, 0.1 percent K HPO 0.01 percent MgSO 7H O, 0.01 percent EXAMPLE 9 FeSO 7H O and 0.01 percent CaCO (sterilization for 60 minutes at 121C) is inoculated with wemgrown Edam If 100 ml. of a nutrient solution according to Example 8 are meyer flasks containing 100 mL of nutrient solution of the 15 inoculated with a spore suspension of the strain Streptomyces same composition, inoculated with strain St 18 and incubated callf?rn'cus Ajrcc and mcubated 90 a at 24C. with stirring and aeration, a culture solution containshakmg f' at a a culture sohmon comammg ing the following CTA units/ml. is obtained: CTA

Duration of Culture CTA Units/ml. 20 EXAMPLE [0 (hours) If 100 ml. of a nutrient solution according to Example 8 are inoculated with a spore suspension of the strain Streptomyces 36 1.9 44 33 gnseus ATCC 10 137 and incubated for 65 hours on a shaking 52 3.8 machine at 23C., a culture solution containing 5.2 CIA units/ml. is obtained.

TABLE 1.DESCRIPTION OF THE STRAINS Strepternyces chrysomallus Strain Lindenbein Type Strain 1 St 4 St 7 St 16 St 18 St 21 St 24 Morphology of the air Sympodially branched Strongly branched sporophores, Sympodially branched bundles with straight or wavy mycelium. bundles with straight or ends strongly spiraled, mostly sporophores; no spirals.

wavy sporophores, no 8-10 turns. spirals. Morphology of the spores... Smooth Smooth Mostly Smooth Smooth. Smooth. Smooth.

smooth, in part with warts. Color of the air myee1ium Only white in the young Mouse grey in Mouse-grey... Only white in the young state or Cream in, See St 1.

state or when poorly the ripe state. when poorly develpart developed, cream with oped, cream with admixtures grey. admixtures of green and of green and grey when copigrey when copiously ously developed. developed. Chromogen formation (see No brown pigment No brown Forms brown N0 brown pigment t No brown pigment.

Table 2, glucose, nutripigment. pigment.

ent, tyrosine-agar, gelatine stab culture.

TABLE 2.BEHAVIO UR OF THE STRAINS ON THE MOST IMPORTANT DIAGNOSTIC NUTRIENTS [G= Growth; AM=Air Mycelium; SP=Soluble Pigment] Streptomyces chrysomallus Lindenbein Type Nutrient Strain St 16 St 18 St 24 Synthetic agar 1 G Strong. crusty, yellow. Strong, crusty, yellow Crusty, colourless Moderate, thin, crusty, colourles AM Dusty, thin, white. Strongly fioury, white to Dusty, later slightly floury, Absent.

to cream. white to cream. SP Pale yellow Yellow Absent Do. Glygggineglycine agar:

G Strong, crusty to Strong, crusty, yellow orange. Crusty, olive tobrown olive. Crusty, yellow to yellow brown.

wrinkly, yellow to yellow orange. AM Du'sltyt indications, Floury,white to cream Dusty, cream, locally grey. Floury, white to cream.

w n e. 7 SP Golden yellow Golden yellow Pale yellow Indications ol yellowish brown. 3

G. Weakly crusty, colourless. AM. Partly fioury, white to cream. SP Absent. "9-. Glucos-asparagineagar: 1

G Good, crusty to pustu- Crusty, yellow Crusty, pale yellowish Crusty, pale yellow.

lar, colorless to pale yellowish. AM indications Dusty, white Floury, whitish to cream Floury to dusty, white.

W l 0. SP Pale yellow- Yellow Pale yellow Pale yellowish. Ca-malate Agar: 1

G Modleratle, finely crusty, Moderate, crusty, pale yellow- Moderate, crusty, colourless Weakly crusty, colourless.

co our ess. AM Indicated, white Absent Absent Absent. SP... 0 Greenish-yellow do Do. Starch plate:

AM Str0ng. crusty, yellow... Crusty, yellowish Crusty, yellow Very thin, crusty, colourless. G Dusty indications, Dusty, locally floury, white Dusty to fioury, white to Indicated, whitish.

white. to cream. cream. SP Absent, starch dcgrada- Pale yellow starch degrada- Absent, starch degradation: Absent, starch degradation:

, i9I lI W-. V .t en owslctate; L ases l k- TABLE 2.-BEHAVIO.UR OF THE STRAINS ON THE MOST IMPORTANT DIAGNOSTIC NUTRIENTS-Conl.

[G=Growth;AM=AirMyccllum;SP=Solublo Pigment] Streptomyces chrysomallus Lindenbein Type Nutrient Strain St 16 St 18 St24 Cellulose broth: 1

G ..0 ..0 o. Glucose-agar: 1

G Strong, crusty to Moderate, crusty, colourloss.. Moderately crusty, colourless Weakly crusty, colourless.

wrinkly, pale yellow. AM Locally dusty, \vhite.... Absellt Absent. SP Yellowish do ..d( lJo. Nutrient, agar: 1

G Good, crusty, yellowish. Slirny, yellow. Crusty, yellowish. Crusty, colourless. AM. Absent Dusty, white to cream... White indications. SP Yellow Yellow.... Yellow Absent. Tyrosine-agar: 3

G Crusty, colourless. Crusty, brownlsh.. Weakly crusty, colourless.

Indicatcd Dusty, white to crcam.. Locally greenlsli yellow. SP Weakly yellowish Yellowish Weakly hrownish Absent. Potato-agar: 4

G Very strong, wrinkly, Strong, crusty, wrinkly, Strong, crusty to wrinkly, Very strong, crusty, yellow golden yellow to orange to olive. yellow green, latcr brown, orange yellow later brownish. brownish. AM Partly present, thinly dusty, Locally dusty, white to Strongly lloury, greenish,

white. greenish yellow. cream or grey. SP Ygllowish, later yellow Yellow olive Greenish brown 4 Green yellow to yellow brown.

rown. Oatflake-agar: 4

G Moderate to good, Thinly cr sty, yellow to Crusty, colourless Crusty, brownish.

crusty, yellow. yellow orange. AM Partly dusty, white to cream.. Floury, white to yellow green. Locally floury, white to greenish.

Yc1l0w Absent Pale yellow to light brown. Litmus milk:

G Ring, colourless Ring, colourless Ring Ring.

Absent Absent Absent Absent.

' Precipitato, no peptonisation Precipltatc, no pept0nisation Precipitatc, no peptonisation. peptonisation. Liiifiers serum nutrient:

G Very strong, wrinkly, Very strong. wrinkly, slimy, Very strong, wrinkly, slimy, Very strong, wrinkly, slimy,

slimy, yellow brown. brown olive. brown olive. olive. AM Absent Absent Absent Absent. SP Abisont, slow liquel'ac' Absent, slow liqueiactiom. Absent, slow liqueiactio Pale yellow, slow liquefaction.

t on. Carrot wedges:

G Strong, crusty to Few colonies, strong, wrinkly, Individual colonies, wrinkly, One colony, crusty to wrinkly,

wrinkly, yellow to olive. brown olive. olive. reddish. AM Dusty indication, white. Locally dusty, white Partly dusty, yellowish Absent. SP Absent Absent Absent Do. Potato wedges:

G Very strong, wrinkly, Very strong, wrinkly, orange Strong, wrinkly brown Very strong, wrinkly.

golden yellow. to bn'ck red. AM Absent Fine as dust, white or Isolated areas, thin white. Floury, white.

yellowish. SP Absent Absent Absent Absent Gelatino-stab culture: 1

G Stgong, crusty, yellow Crusty, yellow Crusty, yellow Wn'nkly, brownish.

rown. AM Absent Dusty, white Dusty, white Flonry to dusty, cream. SP Absent strong liquc- Absent strong liquefaction. Absent, strong liquefaction. Absent, strong liquefaction.

ction. Peptone-iron-agar (Dilco):

G Strong, crusty Strong, crusty. Strong, crusty Strong, crusty. AM" ....0 0 SP Brownish Yellow brown Yellowish brown Ycllowish brown.

1 According to Lindenbein, W. Glucose-agar: KzliPOl instead of 3 According to IIutter, It. KIIZPOI; Ca Mnlatc-Agar: without glyccrinc. 4 According to Wuksman, SA. Potato-agar: Iotatoglucosemgar with- 1 According to you Plotho, see Waksman, S.A. CaCOz and MgSO4.

TABLE 3.GROWTH ON VARI OUS CARBON AND NITRO- TABLE 4 GEN SOURCES [Concentration in the nutrient solution in percent] [Nutrient according to Waksman S.A.: +++=very strong growth; CTA It ++=strong growth; +=rnoderato growth; =l==doubttul growth; 0= 5/ no growth] MgSOofl FQSOAX7 m l, after 4 NaCl KZHPOI H1O CaCO H O days culture Streptomyces chrysomauus 0. 1 0. 01 0. 01 0.01 z. 5 o 2-2 3-2: 88% Carbon source Type Strain St 16 St 18 St 24 8: 01 01 7 d-Xylose 0 l-Arabinoso o gl 8% o. 0 Z Rhamnosc 0 0 02 1 d-Galac o Sucrose :l: 0 M g g 7 W A i Maltese. Maltese/a- Lactose Ramnose 0 3+ T T The fibrinolysokinascs recovered by the process of the in- 0 o o vention are useful as medicines for the known indications. i++ I Literature exists as to the use and dosage [Fletcher et al, J. i o 0 Lab. and Clin. Med. 65, page 713 (I965) In general, it may 35 be stated that this article and others indicate that the product is useful as a thrombolytic agent for the treatment of thromboembolic vascular disease. Dosage rates range from 25,000 +1 to one million CT A units per hour, based on the body size or weight of the patient. Methods for administration and other Bose nutrient O 0 0 0 data are set forth in the article mentioned above.

1 NnNO Instead of (NHmSOt The invention has been described herein with reference to 1 Individual colonies.

.1 1 col0 y certain preferred embodiments. However, the invention is not 9 to be considered as limited thereto as obvious variations will become apparent to those skilled in the art.

What is claimed is:

l. A process for the manufacture of a fibrinolysokinase which comprises growing a culture of the genus Streptomyces, removing proteases by adjusting the pH of the mixture to a pH of about to 4.0, heating the mixture at a temperature of about 50 80C., cooling, adjusting the pH to a pH of about to 10, and recovering the product by fractional precipitation.

2. A process according to claim 1 wherein the pH is initially adjusted to 0 to 4.0 by the addition of a dilute aqueous solution of an inorganic acid, an organic acid, acid salt or mixture thereof.

3. A process according to claim 1 wherein the pH is adjusted to about 5 to after the heating step by the addition of an aqueous solution of an alkali metal or alkaline earth metal hydroxide, oxide, carbonate, bicarbonate or mixture thereof.

4. A process according to claim 3 wherein the fractional precipitation recovery step is carried out after adjustment of the pH to 5 to 10 by dissolving the mixture in an organic solvent, cooling to a temperature of about -40 to 0C., and recovering the product therefrom.

5. A process according to claim 1 wherein the pH of the culture solution is adjusted to a pH of about 1.5 by the addition of about 10 percent aqueous HCI, the mixture is then heated at a temperature of about 65C. for a short period, then cooled, the pH adjusted to about 7.5 by the addition of about 10 per cent aqueous NaOH, the mixture is dissolved in an excess of acetone, cooled and the product recovered therefrom. 

2. A process according to claim 1 wherein the pH is initially adjusted to 0 to 4.0 by the addition of a dilute aqueous solution of an inorganic acid, an organic acid, acid salt or mixture thereof.
 3. A process according to claim 1 wherein the pH is adjusted to about 5 to 10 after the heating step by the addition of an aqueous solution of an alkali metal or alkaline earth metal hydroxide, oxide, carbonate, bicarbonate or mixture thereof.
 4. A process according to claim 3 wherein the fractional precipitation recovery step is carried out after adjustment of the pH to 5 to 10 by dissolving the mixture in an organic solvent, cooling to a temperature of about -40* to 0* C., and recovering the product therefrom.
 5. A process according to claim 1 wherein the pH of the culture solution is adjusted to a pH of about 1.5 by the addition of about 10 percent aqueous HCl, the mixture is then heated at a temperature of about 65* C. for a short period, then cooled, the pH adjusted to about 7.5 by the addition of about 10 percent aqueous NaOH, the mixture is dissolved in an excess of acetone, cooled and the product recovered therefrom. 